Active-Site-Matched Fluorescent Probes for Rapid and Direct Detection of Vicinal-Sulfydryl-Containing Peptides/Proteins in Living Cells

Vicinal-sulfydryl-containing peptides/proteins (VSPPs) play a crucial role in human pathologies. Fluorescent probes that are capable of detecting intracellular VSPPs in vivo would be useful tools to explore the mechanisms of some diseases. In this study, by regulating the spatial separation of two maleimide groups in a fluorescent dye to match that of two active cysteine residues contained in the conserved amino acid sequence (-CGPC-) of human thioredoxin, two active-site-matched fluorescent probes, o-Dm-Ac and m-Dm-Ac, were developed for real-time imaging of VSPPs in living cells. As a result, the two probes can rapidly respond to small peptide models and reduced proteins, such as WCGPCK (W-6), WCGGPCK (W-7), and WCGGGPCK (W-8), reduced bovine serum albumin (rBSA), and reduced thioredoxin (rTrx). Moreover, o-Dm-Ac displays a higher binding sensitivity with the above-mentioned peptides and proteins, especially with W-7 and rTrx. Furthermore, o-Dm-Ac was successfully used to rapidly and directly detect VSPPs both in vitro and in living cells. Thus, a novel probe-design strategy was proposed and the synthesized probe applied successfully in imaging of target proteins in situ.